D Domains are Affinity Matured Through Mutagenesis

A61EA61K

G8A

I59AI59Y

K55EK55Y

N66R R17AR17E

V62EV62R

W16A

E58A

W16T

104

105

106

104 103 102 101

koff (1/s)

k on

(1/M

s)

050100150200

KD (nM)

■■ A CD123-binding D domain was subjected to mutagenesis to identify point mutations that could either increase or decrease binding affinity■■ A homology model of the scaffold illustrates the location of representative mutations in red (left)■■ D domain mutants were expressed as monovalent MBP-fusions and binding kinetics were assessed by surface plasmon resonance (right)■■ KD values for binding of CD123-HIS to D domains spanned a 150-fold range (1.4 nM to 212 nM), with off-rate contributing more to the distribution than on-rate, suggesting these mutations impact stabilization of the complex, once formed

sparX Incorporating High Affinity D Domain Enhances Potency of ARC-T Cells

-2 -1 0 1 20

1x106

5x105

0

1x106

5x105

Cell

Lysi

s(L

ucif

eras

e Si

gnal

)

Cell

Lysi

s(L

ucif

eras

e Si

gnal

)MOLM13 MOLM13(CD123-/-)

MOLM13 MOLM13

-2 -1 0 1 2

-2 -1 0 1 2

0.00

0.25

0.50

0.75

1.00

sparX Concentration (Log10, nM) sparX Concentration (Log10, nM)

sparX Concentration (Log10, nM) sparX Concentration (Log10, nM)

IL-2

(ng/

mL)

sparX-α3DsparX-cg77sparX-cg77.1

-2 -1 0 1 2

0.0

1.0

2.0

3.0

4.0

5.0

IFN

(ng/

mL)

Monovalent

DiseasedCell

ARC-T

■■ Monovalent sparX were generated with the cg77 D domain or a high-affinity variant (sparX-cg77.1), with an approximately 10-fold lower KD, as measured by SPR■■ As measured by luciferase signal, ARC-T cell mediated lysis of AML-derived MOLM13/luciferase cells requires CD123-binding sparX (top left), CD123 expression on target cells (top right) and ARC-T cells (not shown)■■ ARC-T cell mediated target cell lysis (top left), IL-2 (bottom left) release and IFN release (bottom right) are dose and affinity dependent ■■ As measured by EC50 and as compared to the parental sparX-cg77 the high-affinity variant (sparX-cg77.1) demonstrated enhanced potency for target cell lysis (4-fold increase), IL-2 release (5-fold increase) and IFN release (6-fold increase)

Combinations of Affinity and Valency of D Domain Can Be Used to Further Broaden Dynamic Range of sparX

Bivalent DiseasedCell

ARC-T

sparX-α3D

sparX-cg77sparX-cg77(HA)

sparX-cg77(LA)

Monovalent DiseasedCell

ARC-T

sparX-cg77(LA)sparX-cg77sparX-cg77(HA)

-3 -2 -1 0 1 2

0

50

100

sparX Concentration (Log10, nM)

% L

ysis

■■ sparX were generated in both mono and bivalent formats, utilizing the parent cg77 D domain or variants incorporating high affinity (HA) or low affinity (LA) mutations■■ ARC-T cell mediated lysis of CD123-expressing, NALM6 cells is modulated by CD123-binding sparX■■ The extent of target cell lysis is dose-dependent for each sparX and collectively the EC50s span 3 logs■■ Low affinity sparX manifest greatest gain in potency when formatted as a bivalent ■■ The high affinity, monovalent sparX (sparX-cg77 [HA]) outperforms the bivalent sparX-cg77

Chimeric Antigen Receptors Incorporating Novel (non-scFv) Binding Domains Targeting CD123 Direct Potent Antitumor Activity of T Cells: Correlation Between Affinity and Activity

David W. LaFleur, Justin P. Edwards, Liubov Zaritskaya, Ankit Gupta, Haiying Qin, Terry J. Fry, Jeffery S. Swers, C. Jenny Mu, Laura K. Richman, David M. Hilbert

Arcellx, Inc., Gaithersburg, MD

Abstract 3243

Results

CAR T Cells Incorporating D Domains Control AML Tumor Growth In Vivo

α3D-CAR

CAR-T

1.0 x 1060.8 0.6 0.4 0.2

Luminescence

Day 0

Day 7

Day 14

Day 21

Day 28

Day 35

Day 42

Anti-FLAG/CAR0 103 104 105

CAR+87.8

CD123scFv-CAR

0 103 104 105

CAR+86.7

cg77-CAR

0 103 104 105

CAR+90.6

■■ A CD123-binding D domain was identified through phage display methods and further deimmunized to remove predicted T cell epitopes (cg77)1 ■■ Detection of N-terminal Flag epitope by flow cytometry demonstrated comparable percent positive CAR expression on T cells between non-binding D domain CAR (a3D-CAR), cg77-CAR and a 32716-scFv-based CAR targeting CD123 (CD123scFv-CAR)■■ AML-derived, CD123-positive MOLM14 tumor cells (3x106) were administered systemically 1 day prior to administration of 3x106 a3D-CAR, cg77-CAR and CD123scFv-CAR transduced primary human T cells■■ Both cg77-CAR and CD123scFv-CAR eliminated detectible levels of tumor cells as measured by IVIS imaging

ARC-sparX Platform Incorporating D Domains Control AML Tumor Growth In Vivo

1.0 x 1060.8 0.6 0.4 0.2

Luminescence

sparX-cg77 Bivalent

0.5 mg/kg 0.05 mg/kg

sparX-α3D5 mg/kg

ARC-T Cells5×106

cg77-CAR5×106

Day 0

Day 7

Day 14

Day 20

Day 27

Day 31

1 x 106

MOLM-145 x 106

CAR + T-cells +/- sparX

0-2

Once daily sparX

10 21

■■ cg77 was incorporated as a conventionally formatted CAR (cg77-CAR) or bivalent sparX (sparX-cg77 Bivalent)■■ ARC-T cell mediated anti-tumor activity was sparX-cg77 dose dependent■■ To achieve greater tumor eradication, alternative sparX dosing and/or CD123 binding affinity variants may be required — and were explored

Introduction

D Domains Constitute a Robust Scaffold for Rapid Development of Targeting Domains

25C40C

55C70C

100C

1 2 3 4 5

0

1

2

3

4

Dilution (log)

Targ

et B

indi

ng (A

450)

D domain

cg09

cg25

cg24

cg34

cg18

cg22

cg05

cg04

cg07

cg26cg39

cg45

cg33

cg08

cg20

cg38

cg56

cg53

cg28

cg54

cg11

cg14

cg40

cg44

cg21

cg52

cg12

cg51

cg19

cg16

cg03

cg10

cg43

cg46

cg01

cg06

cg17

cg37

cg41

cg49

cg13

cg55

cg42

cg48

cg23

cg36

cg50

cg30

cg27

cg47cg35

cg32

cg02

cg31

1 2 3 4 5

0

1

2

3

4

Dilution (log)

Targ

et B

indi

ng (A

450)

scFv

Thermal Stability Library Diversity In Silico Assessment

0.0

0.5

1.0

1.5

2.0

0.0 0.5 1.0 1.5 2.0Control Binding (A450)

Targ

et B

indi

ng (A

450)

-26

-24

-22

-20

Wimley

0.0

0.5

1.0

1.5

2.0

0.0 0.5 1.0 1.5 2.0Control Binding (A450)

Targ

et B

indi

ng (A

450)

2

3

4

5

6Epitope

0.0

0.5

1.0

1.5

2.0

0.0 0.5 1.0 1.5 2.0Control Binding (A450)

Targ

et B

indi

ng (A

450)

123456

Count

Immunogenicity

Sequence Frequency

Hydrophobicity

■■ Conventional chimeric antigen receptors utilize an antibody-derived scFv as the targeting domain■■ D domains are 73 aa in length (approximately 1/3 the size of a scFv) and contain no disulfide bonds or native glycosylation1

■■ Rapid folding of the scaffold2 imparts exceptional thermal stability (left) while simultaneously accommodating a high number of residue substitutions■■ Multiple library designs of the triple alpha helical structure, translate into a high degree of paratope and sequence diversity (center)■■ Combined in vitro and in silico screens of phage library clones afford predictive assessments of D domain target specificity, sequence diversity, expression and immunogenicity (right)■■ D domains are selected for low potential immunogenicity and are ultimately engineered to produce an Abzena EpiScreen™️ score of zero3

The ARC-sparX Platform Conventional CAR-T cell therapies often target tumors through a mono-specific receptor that is constitutively expressed. To improve the flexibility and control of T-cell response, we developed the ARC-sparX platform, which separates the antigen recognition and killing functions of the conventional CAR-T cell.

The ARC-sparX platform is comprised of two key components:1. sparX (soluble protein antigen-receptor X-linker) protein: binds specific

antigens on diseased cells and flags those cells for destruction. 2. ARC-T Cells (Antigen Receptor Complex T Cells): bind sparX proteins and kill

flagged cells. Tri-complex (ARC-T + sparX + target cell) must be formed to activate cytolytic killing.

ARC-T

DiseasedCell

sparX

TAG

sparX protein■■ No inherent activity■■ Mono-valent affinity comparable to that of scFv■■ Engineered to minimize immunogenic potential■■ “TAG” is a fragment of human alpha fetoprotein (AFP)

ARC-T Cell■■ Conventional CAR architecture incorporating an anti-TAG D domain■■ Receptor for “TAG” has affinity in low nanomolar range■■ Only activated upon formation of tri-complex (ARC-T + sparX + antigen-expressing target cell)■■ Same viral vector regardless of antigen target

sparX Can Be Mono-valent, Multi-valent, or Multi-specific

sparX #1 sparX #2

DiseasedCell

ARC-T ARC-T

SimultaneoussparX

DiseasedCell

ARC-T

Bi-specificsparX

Bi-valentsparX

DiseasedCell

ARC-T

■■ Can be given in combination or in sequence■■ Addresses low density or heterogeneous expression of antigen■■ Bi-specific sparX proteins support “AND-gated” as well as “OR-gated” targeting of diseased cells

Presented at the American Association for Cancer Research 2020 Virtual Annual Meeting II, June 22-24, 2020

DLaFleur@arcellx.com

©2020 Arcellx, Inc. All rights reserved.

Monovalent, High-Affinity CD123 sparX Eliminates Detectable AML Tumors In Vivo

Day 28

sparX-cg77(HA) – monovalent

3 mg/kg 0.3 mg/kg1 mg/kg

sparX-α3D3 mg/kg

cg77(HA)-CAR

Day 0

Day 7

Day 14

Day 21

Day 4

1.0 x 1060.8 0.6 0.4 0.2

Luminescence

1 x 106

MOLM-145 x 106

ARC-T or CAR-T

0-2

30

Every other day sparX

10 20

■■ A high affinity CD123 D domain is incorporated into monovalent sparX (sparX-cg77[HA]) and a conventionally formatted CAR (cg77[HA]-CAR)■■ In vivo anti-tumor activity of MOLM14 tumors by ARC-T is dependent on sparX dose■■ Cohorts receiving 1 and 3 mg/kg sparX-cg77(HA), every other day, achieved comparable anti-tumor activity to that of the conventionally formatted CAR

Conclusions ■ As exemplified by D domains discovered and optimized to bind CD123, D domains constitute a robust scaffold for development of therapeutic targeting domains

■ CAR T cells comprised of D domains targeting CD123, direct potent in vivo anti-tumor activity of AML-derived tumors comparable to that of a conventional, scFv-based CAR

■ The ARC-sparX platform affords the ability to modulate T-cell activity by controlling the specificity, dose, affinity and valency of the sparX protein

■ sparX proteins targeting CD123, in combination with ARC-T cells, inhibit the growth of AML-derived tumors in vivo and may offer a novel therapeutic approach for treating AML

■ More broadly, the ARC-sparX platform affords an opportunity for treating other challenging cell therapy indications where greater T-cell control may be warranted

References1. Qin, Haiying, et al. “Chimeric antigen receptors incorporating D domains targeting CD123 direct potent mono-

and bi- specific antitumor activity of T cells.” Molecular Therapy 27.7 (2019): 1262-1274. 2. Zhu Y, Alonso DO, Maki K, et al. Ultrafast folding of alpha3D: a de novo designed three-helix bundle protein.

Proc Natl Acad Sci U S A. 003;100(26):15486-15491. doi:10.1073/pnas.2136623100. 3. Baker MP, Jones TD. Identification and removal of immunogenicity in therapeutic proteins.

Curr Opin Drug Discov Devel. 2007;10(2): 219-227.